Recent measurements of acetylcholine (ACh) release from motor nerves demonstrate that cholinergic drugs alter ACh release. The goal of this project is to determine the significance of such findings. One possibility is that motor nerve endings have ACh receptors (AChRs) that modulate ACh release. However, biochemical and radiolabel assays have led to different conclusions concerning whether there is positive or negative feedback of ACh release and whether muscarinic as well as nicotinic AchRs are involved. One aim of this project is to make a direct comparison of the results and limitations of the biochemical and radiolabel assays for ACh release. A second aim is to test additional combinations of cholinergic drugs to define the nature of the (putative) receptor(s) modulating ACh release. Most important is the determination of the relevance of these cholinergic drug actions on ACh release to the physiology and clinical pharmacology of the neuromuscular junction. Thus, using rodent phrenic nerve-diaphragm preparations, the actions of drugs on ACh release will be compared to their postjunctional actions measured under similar conditions, comparisons which have never been done before. Changes in ACh release will be related to changes in either force of contraction or endplate potentials. Additionally, changes in ACh release as determined from direct measurements of ACh will be compared to electrophysiological estimates, which are used far more commonly. In the clinical use of neuromuscular drugs, it is now commonly assumed that tetanic fade and train-of-four fade are due to the actions of neuromuscular blocking drugs to decrease ACh release, while the use of muscarinic antagonists is thought not to significantly alter neuromuscular transmission. Along with providing new information on the pharmacology of ACh release, comparing methods of ACh assay and relating changes in ACh release to postjunctional changes, this project will provide new insights on these clinical assumptions.